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Introduction

Stroke, including local and widespread infarction, is a common cause of mortality and disability in patients with brain ischemia in developed and developing countries (1). The major cause of stroke is cerebral artery occlusion due to local thrombosis ensuing following the rupture of carotid atherosclerotic plaques (CAP) (2,3). Among the various causes that influence the destabilization and rupture of atherosclerotic plaques, inflammatory reactions take an important place (4). The mRNA expression of numerous genes involved in inflammatory reactions is also altered in atherosclerotic plaques (5,6).

The kinins, as a family of inflammatory peptides, are involved in a variety of physical processes, including vasodilation and vascular permeability (7). During inflammatory conditions, kinins induced vasodilation and pain, increased vascular permeability and recruited inflammatory cells to the sites of injury by activating specific receptors, B1 and B2 (8). B1 receptors (B1R) are weakly expressed under normal physiological circumstances, but their expression could be increased markedly by inflammation and injury (9). By contrast, B2 receptors (B2R) are expressed consecutively in healthy and pathological tissues and could also be enhanced in the inflammatory processes (8). Activated kinin receptors increased the production of endothelial nitric oxide (NO) and prostaglandins by enabling NO synthase and phospholipase A2 in endothelial cells. NO and prostaglandins act on the smooth muscle, leading to vasodilation, increased permeability and pain. In addition, kinins also interacted directly with its receptors located in vascular smooth muscle cells, resulting in the activation of several enzymes, such as mitogen-activated protein kinases and protein kinase C, accompanied with the generation of reactive oxygen species, cellular migration and production of extracellular matrix proteins (10).

Previously, a number of studies have shown the increased expression of B1R and B2R in atherosclerotic vessels and peripheral blood cells of patients with atherosclerosis (11,12). Particularly, in the cardiovascular systems, kinins have been found to be involved in the pathogenesis of heart failure (13). Notably, whereas kinins promoted angiogenesis, they also inhibited post-angioplasty restenosis (14). However, despite the extensive studies focused on the kinin-mediated inflammation in various pathologies, little is known regarding their performance in the formation and destabilization of human CAP.

In the present study, the expression of the genes encoding kinin receptors in human CAPs were assessed and compared to arteries obtained from normal controls, to improve the understanding of the role of kinin-mediated inflammation in the formation of human CAP, and therefore develop new preventive strategies. In addition, the plaques were classified into two groups based on the presence of surface ruptures, so as to further assess the performance of the kinin receptors in the destabilization of atherosclerotic plaques.

Patients and methods

Patients

Carotid plaque specimens from 47 patients undergoing carotid endarterectomy for symptomatic (transient ischemic attacks and minor strokes) or asymptomatic stenosis >70%, as confirmed by magnetic resonance angiography (MRA) or conventional arteriography, were collected. Each patient was subjected to a detailed history assessment and a physical examination, including routine blood biochemistry tests, electrocardiography, chest radiography, ultrasound cardiograph, carotid contrast-enhanced magnetic resonance imaging, computed tomography, magnetic resonance imaging and MRA of the brain. The vascular risk factors, as well as previous antihypertensive, statin and antiplatelet treatments, were also recorded (Table I). A total of 10 inferior mesenteric arteries dissected from colectomy specimens following elective surgery were used as controls. Each control patient was assessed to exclude symptomatic atherosclerotic disease by history and examination.

Table I. Demographic and clinical data of patients and controls.

Table I.

Demographic and clinical data of patients and controls.

Variables	Patients	Controls	P-value
Number	47	10
Age, years	56.1±7.3	55.9±10.1	NS
Gender, male/female	33/14	7/3	NS
Smoking	26	4	NS
Diabetes	12	1	NS
Hypertension	30	3	NS
Coronary artery disease	14	0	NS
Family of IHD	22	2	NS
Aspirin	19	1	NS
Clopidogrel	2	0	NS
Warfarin	4	0	NS
Statins	14	0	NS

[i] IHD, ischemic heart disease; NS, not significant.

The study was approved by the Medical Ethics Committee of The Fifth Affiliated Hospital of Zhengzhou University (Zhengzhou, China). Written informed consent was obtained from each participant.

Specimens

The endarterectomy specimens included the atheromatous plaque, the adjacent intima and the medial layers. The control arteries were full-thickness and consisted of the adventitial layer. Plaques and control arteries were frozen immediately in liquid nitrogen and stored at −80˚C until further processing. Based on the presence of surface ruptures on plaques, the specimens were assigned into two groups: The stable plaque group, which contained a diffuse intimal thickening, a small eccentric plaque or a necrotic core surrounded by fibrous tissue without surface ruptures; and the unstable plaque group, which contained complex plaques with a possible surface defect, hemorrhage or thrombus.

RNA extraction

Total RNA was extracted from freshly isolated pulverized frozen tissue (∼25 mg) with TRIzol® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) following the manufacturer's instructions and was solubilized in RNAase-free water. All the extracts were treated with DNase to avoid contamination from genomic DNA. Final concentration and purity were qualitatively evaluated by electrophoresis in 1% agarose gel stained with ethidium bromide and quantified spectrophotometrically (Nanodrop DA-2000; Thermo Fisher Scientific, Wilmington, DE, USA).

Primers

The primers and probes for amplification of B1R and B2R were determined using the Primer Premier 6.0 computer program (Premier Biosoft, Palo Alto, CA, USA) and their sequence homologies were checked using the GenBank database (www.ncbi.nlm.nih.gov/genbank).

The primer sequences used were as follows: i) B1R forward, 5′-ACGATTCTCCCACCTCA-3′; and reverse, 5′-AGCCCA AGACAAACACCA-3′; and ii) B2R forward, 5′-CTACCC AGCCCTTGAAAGAT-3′; and reverse, 5′-GAAGACGCT GAGGACAAAGAT-3′.

The β-actin gene was used as the endogenous control and amplified simultaneously with the target gene. The primer sequences used for β-actin were as follows: Forward, 5′-AAG GAAGGCTGGAAGAGTGC-3′; and reverse, 5′-CTGGGA CGACATGGAGAAAA-3′.

Reverse transcription-polymerase chain reaction (RT-PCR)

The mRNA level was determined by RT-PCR. The first-strand cDNA was synthesized from 2 µg total RNA with PrimeScript Reverse Transcriptase (Takara Bio, Inc., Shiga, Japan). PCR was carried out by incubating each cDNA sample with primers (0.5 µM each), Blend Taq® polymerase (1.25 U; Takara Bio, Inc.) and a deoxynucleotide. The cycling conditions were as follows: RT, 42˚C for 60 min; polymerase activation, 70˚C for 15 min; PCR, 35 cycles at 94˚C for 3 min and 94˚C for 30 sec; and final elongation, 72˚C for 40 sec. Reactions were performed in triplicate and in a total volume of 25 µl. PCR products were electrophoresed in 1% agarose gels. Bands were stained with ethidium bromide (Sigma, St. Louis, MO, USA) and detected with an LAS-3000 mini CCD camera (FUJIFILM Investment Co., Ltd., Shanghai, China). The Eagle Eye II analyzer (Strategene, La Jolla, CA, USA) was used to determine the optical density (OD) of the target and reference mRNA. The mRNA level was recorded as the relative value of the OD of the target gene to that of β-actin.

Statistical analysis

Results are presented as mean ± standard error of the mean. Statistical analysis was performed with SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). Gender differences between patients and control groups were assessed using the χ2 test. Comparisons in the B1R and B2R mRNA levels across different groups of patients and controls were performed using analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

Results

The demographic and clinical data of the patients and controls are shown in the Table. I. There was no significant difference between patients and controls in gender, symptomatic carotid disease, individual or combined risk factors, treatment or coexistence of other vascular disease (Table. I).

One specimen was collected from each patient and 47 plaques were obtained in total. These specimens were classified into two groups respectively, based on the presence of surface ruptures on plaques: i) The stable plaque group with no defects on the surface (n=28); and ii) the unstable plaque group with surface defects or ruptures (n=19).

Increased expression of the genes encoding B1R was observed in the stable and unstable plaque groups relative to the control group (0.98±0.37 and 1.69±0.65 vs. 0.07±0.06). The B1R mRNA expression was significantly increased in the plaque groups as compared to the control group (Fig. 1). When comparing the two plaque groups, the relative ratios of B1R to β-actin in the mRNA level in the unstable plaque group were notably higher than that in the stable plaque group (Fig. 1).

Figure 1. (A) Polymerase chain reaction results for β-actin and B1R in the stable plaque group (SPG), unstable plaque group (UPG) and control group. (B) Differences in the relative ratios of B1R to β-actin in the mRNA expressions across the different groups. *P<0.05; **P<0.05.

The expression of the genes encoding B2R were also increased in the stable and unstable plaque groups as compared to that in the control group (0.98±0.43 and 1.16±0.52 vs. 0.48±0.16). The differences in the relative ratios of B2R to β-actin in the mRNA expression between the plaque groups and control group were significant (Fig. 2). When the two plaque groups were compared, no notable difference was identified in the relative ratios of B2R to β-actin in the mRNA expression.

Figure 2. (A) Polymerase chain reaction results for β-actin and B2R in the stable plaque group (SPG), unstable plaque group (UPG) and control group. (B) Differences in the relative ratios of B2R to β-actin in the mRNA expressions across the different groups. #P<0.05; *P<0.05.

Discussion

In the present study, the B1R and B2R mRNA expression in human CAPs was significantly unregulated as compared to control arteries, suggesting that kinin-mediated inflammation contributes to the formation of atherosclerotic plaques, which is in accordance with previous findings showing that kinin receptors were increased in the cardiovascular and peripheral vessel atherosclerosis (4,15,16). When the plaques were classified into two groups based on the presence of surface ruptures, the relative ratios of B1R to β-actin in the mRNA expression in the unstable plaque group were higher than that in the stable plaque group. However, no significant differences were observed in the relative ratios of B2R to β-actin in the mRNA levels between the stable and unstable plaque groups. These findings may indicate that the B1R gene expression contributes to the destabilization or rupture of human CAP. To the best of our knowledge, similar studies focusing on the role of kinin-mediated inflammation in the destabilization of human CAP have not been reported previously.

Traditionally, it is believed that the formation of atherosclerotic plaques simply reflects the deposition of lipids within the vessel wall of medium-sized and large arteries (15,16). Currently, it is recognized that inflammation, including focal and systematic inflammation, are important players in the atherosclerotic processes (15). As important inflammation members, the kinin systems are also involved in the regulation of vasomotor, endogenous fibrinolysis in humans and their biological effects are mediated by two G protein-coupled receptors, B1 and B2 (7,15). As stated, B2R is constitutively expressed in the majority of tissues under normal conditions. In the cardiovascular system, B2R is mainly located on the surface of endothelial cells and inflammatory cells, including monocytes, which can promote the vasodilation and fibrinolysis by acting through NO and prostaglandin (8). In contrast to B2R, B1R is hardly expressed under physical conditions, but its expression could be markedly increased under inflammatory conditions, which triggers the release of numerous inflammatory mediators, including tumor necrosis factors-α and interleukins (17,18). The activated monocytes have a higher expression of B1R, but the B2R activity tends to decrease (19). Dabek et al (4) reported a higher increase in the B1R gene expression in the mononuclear cells from patients with atherosclerosis. The dysfunction of endothelial cells and monocyte infiltration are two important contributors in the formation of atherosclerotic plaques, particularly for those with surface ruptures, which results in the accumulation of monocytes and dysfunctional endothelial cells accompanied with the subsequent upregulation of B1R and B2R (16). Consistent with prior findings, our present study showed the notable increased expression of B1R and B2R mRNA in human CAPs as compared to controls, providing evidence for the hypothesis that kinin-mediated inflammation contributes to the formation of human CAP.

Previous studies have shown that the instability of atherosclerotic plaques in the cycling system is in part determined by focal factors, but systemic factors such as inflammation and infections may also be extremely important components (20). In particular, the acute ischemic events have been reported to be more closely associated with the morphological features rather than the number of plaques or degree of stenosis (20,21). Pathological studies of endarterectomy specimens demonstrated the plaque ulceration and rupture as the morphological features of previously symptomatic stenosis compared to asymptomatic plaques (21–23). Further comparison between symptomatic and asymptomatic endarterectomy specimens showed that the plaque areas covered by inflammatory cells were notably larger in symptomatic CAPs than asymptomatic ones, suggesting the involvement of inflammation in the plaque destabilization (16,24). In the present study, the B1R gene expression was significantly upregulated in the unstable plaques relative to stable ones, indicating that kinin-mediated inflammation plays an important role in the instability of human CAPs via B1R. Such a finding also provides useful information for developing novel therapies targeted at stabilizing the atherosclerotic plaques based on the systematic inflammation.

As mentioned above, kinin receptors are widely expressed in the endothelial cells and monocytes in the peripheral blood in the cycling systems. Apart from their role in the formation of human atherosclerotic plaques (19), the kinin receptors also play an important role in the instability of the plaques in the cardiovascular systems and carotid artery (15,25,26), making them ideal targets for preventing and treating systematic atherosclerosis. Furthermore, it has been demonstrated on experimental models that the blockage of B1R may reduce the systematic inflammatory response and therefore prevent the formation of atherosclerotic plaques (27). The impacts of kinin-mediated inflammation on the development of atherosclerotic plaques and their destabilization may open new avenues for future treatments.

However, the inflammation reactions mediated by kinins in the formation of human CAP are complex processes that involve numerous molecules and multiple signal pathways. The present study showed the involvement of the kinin-mediated inflammation in the formation and destabilization of human CAP on the mRNA level, but did not determine the subsequent protein expressions following translation as they are beyond the focus of the present study. However, the subsequent signaling pathways following receptor expression may also be quite complex processes, which require further investigation (10,11). In addition, the blockage of kinin receptor expression may prevent the formation of CAPs, a hypothesis that should be tested in future studies.

In conclusion, the present study demonstrated that the patients with CAP experienced notable alterations in the kinin receptor expression at the gene level. The significant upregulation of B1R and B2R mRNA expression in human CAPs revealed that the kinin-mediated inflammation played an important role in the atherosclerotic processes and also disclosed them as novel therapeutic targets for preventing the formation of atherosclerotic plaques. The unstable atherosclerotic plaques exhibited a higher ratio of B1R to β-actin in mRNA levels, suggesting the increased B1R mRNA as a potential target for reducing the destabilization of plaques. Future studies are required to explore the precise mechanism of inflammation causing the development and destabilization of atherosclerotic plaques and develop novel specific therapeutics targeted at such a mechanism.

Acknowledgements

The present study was supported by grants from the Zhengzhou Committee of Science and Technology (no. 083SGY2612-9).

References